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During dynamic exercise, the adenosine triphosphate (ATP) demand increases significantly and its replacement occurs through the glycogen and fatty acids catabolism. The effects of exercise on intramuscular glycogen stores have been widely investigated. However, there is no information about ultra endurance exercise effects on muscle metabolism. In last decades 5’ adenosine monophosphate-activated protein kinase (AMPK) protein has been described as a sensor of changes in energy sources, being directly affected by muscle contraction. The aim of this study was to evaluate the effects of ultra endurance exercise on muscle glycogen (GM) stores and the expression of AMPK protein. Methods: twenty four male Wistar rats, were randomly divided into four groups (sedentary S= 6, sedentary exhaustion SE = 6, trained T =6, and trained exhaustion TE = 6), where T and TE groups run progressively on a treadmill 5d/wk for 12 weeks (60% Vmáx). At the end of training period SE and TE were submitted to an ultra-endurance exercise (60% Vmax) until exhaustion. The gastrocnemius of both legs were excised for glycogen analysis by colorimetric and by optical density method. Gene and protein AMPK expression was determined by real time RT-PCR and western blotting respectively. Results: SE and TE groups showed significant depletion of the GM storages (S = 0.213 ± 0.003, T = 0.203 ± 0.007, SE = 0.031 ± 0.006, TE = 0.028 ± 0.006 mg / g protein) (p <0.001). The liver glycogen was significantly lower in TE when compared to T group (p <0.05 ) (S = 1.157 ± 0.09, SE = 1.002 ± 0.05, T = 1.533 ± 0.05 and T = 1.037 ± 0.05). A significant decrease in plasma glucose was observed in TE group (p <0.05) (S = 90.43 ± 2.47, SE = 85.75 ± 3.18, T = 84.84 ± 2.19, and 76.02 ± TE = 5.60). The AMPK protein expression increased in T and TE groups (p <0.05). However, gene expression increased only in TE group (p <0.05). Conclusion: The ultra-endurance exercise depletes the GM reserves predominantly in type I muscle fibers. The training alone affected positively the protein expression of AMPK. This change was more evident when considered the exhaustion factor.